CN114392256B - Application of cyanidin in prevention and treatment of vascular calcification - Google Patents

Abstract

The invention belongs to the technical field of medicine, and specifically relates to the application of cyanidin in the prevention and treatment of vascular calcification. In order to find a new compound for the treatment of VC and provide a better prevention and treatment plan for VC patients, the invention discloses cyanidin Application in the prevention and treatment of vascular calcification, through the treatment of cyanidin in high Pi-induced calcified male mice and HASMC-induced calcification, it was found that cyanidin can relieve vascular calcification, and it is expected to be applied to the prevention and treatment of vascular calcification diseases among. The invention not only provides a new application of cyanidin, but also provides a new idea for treating vascular calcification.

Description

Application of cyanidin in prevention and treatment of vascular calcification

technical field

The invention belongs to the technical field of medicine, and in particular relates to the application of cyanidin in preventing and treating vascular calcification.

Background technique

Chronic kidney disease (CKD) is an important public health problem, with 697.5 million CKD patients worldwide in 2017. CKD has caused 1.2 million deaths worldwide and is expected to increase to 2.2 million by 2040. Cardiovascular disease is the main cause of early death in CKD patients, and vascular calcification (VC) is an important culprit. But there is still no effective treatment for VC. Therefore, there is a need for new compounds to be used in the treatment of VC to provide better prevention and treatment options for VC patients.

Studies have shown that the bioactive substances of flavonoids, especially anthocyanins, have health benefits. Anthocyanins are composed of cyanidin, peonidin, delphinidin, pelargonidin, pemnidin and malvidin. Glycoside (aglycone)-based. Anthocyanins have two hydroxyl groups on the B ring and are the most widely distributed pigments in plants. Among them, the most representative anthocyanin in edible plants is cyanidin, followed by delphinidin, geranium and paeoniflorin. Studies have shown that anthocyanins can resist oxidation, anti-inflammation, anti-tumor, anti-atherosclerosis, anti-aging, anti-diabetes and increase the bioavailability of endothelial-derived nitric oxide, etc. In a meta-analysis of six studies, the association between higher anthocyanin intake and reduced risk of all-cause mortality was primarily due to a reduced risk of cardiovascular death. However, there is no report about cyanidin alleviating VC.

Contents of the invention

In order to overcome the deficiencies in the prior art above, the present invention proposes the application of cyanidin in the prevention and treatment of vascular calcification. The present invention has found that cyanidin has the function of alleviating vascular calcification. The present invention not only provides cyanidin It also provides a new idea for the treatment of vascular calcification.

In order to achieve the above object, the technical solution adopted in the present invention is:

The invention provides the application of cyanidin in the preparation of medicine for preventing and treating vascular calcification.

Preferably, the vascular calcification is intimal calcification. Therefore, cyanidin can be used in the prevention and treatment of vascular calcification chronic kidney disease.

The invention also provides the application of cyanidin in the preparation of medicine for alleviating vascular cell calcification.

Preferably, said vascular cells comprise vascular smooth muscle cells.

Preferably, the cyanidin is chlorinated cyanidin, its molecular formula is: C ₁₅ H ₁₁ ClO ₆ , and its structural formula is as follows:

The content of cyanidin in foods such as black rice is relatively high, and the present invention finds through animal and cell experiments that chlorinated cyanidin has a therapeutic effect on the calcification of C57BL/6 mice induced by high Pi, and has a therapeutic effect on human vascular smooth muscle cells. (human vascular smooth muscle cells, HASMC) also proved to have a relieving effect. It is expected to apply cyanidin chloride in the treatment of VC, that is, the present invention discovers a new application of cyanidin chloride—prevention and treatment of VC.

The present invention also provides a medicine for preventing and treating vascular calcification or a medicine for alleviating vascular cell calcification, the medicine comprising cyanidin.

Preferably, the drug also includes a drug component that can have a synergistic effect with cyanidin.

Preferably, in order to enrich the application form of the drug so that it can be used in different ranges, the drug also includes a pharmaceutically acceptable carrier.

Further, the carrier is an acceptable functional pharmaceutical excipient in the pharmaceutical field, including surfactants, suspending agents, emulsifiers, and some new pharmaceutical polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid polylactic acid copolymer (PLGA), hyaluronic acid, etc. Excipients such as diluents, binders, lubricants, disintegrants, solubilizers, and stabilizers may also be included.

Preferably, in order to improve the scope of application of the medicine, the medicine includes tablets, capsules, granules, dripping pills, liquids and injections. Pharmaceutical formulations can be administered orally or parenterally (eg intravenously, subcutaneously, intraperitoneally or topically), and if certain drugs are unstable under gastric conditions, they can be prepared as enteric-coated tablets.

Compared with prior art, the beneficial effect of the present invention is:

In order to find new compounds for the treatment of VC and provide a better prevention and treatment plan for VC patients, the present invention discloses the application of cyanidin in the prevention and treatment of vascular calcification, through the induction of calcification in high Pi-induced male mice and HASMC During the process of calcification, cyanidin was treated, and it was found that cyanidin can relieve vascular calcification, and it is expected to be applied to the prevention and treatment of vascular calcification diseases. The invention not only provides a new application of cyanidin, but also provides a new idea for treating vascular calcification.

Description of drawings

Fig. 1 is the toxic effect of different concentrations of cyanidin on HASMC after 24 hours (a) and 48 hours (b);

Fig. 2 is the alleviating effect of cyanidin on HASMC calcification;

Figure 3 is the effect of cyanidin on the body weight of C57BL/6 male mice after high-phosphorus diet-induced calcification;

Figure 4 shows the staining results of cyanidin on the aorta of C57BL/6 male mice after high-phosphorus diet-induced calcification.

Detailed ways

Specific embodiments of the present invention will be further described below. It should be noted here that the descriptions of these embodiments are used to help understand the present invention, but are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not constitute a conflict with each other.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and the test materials used in the following examples, unless otherwise specified, can be purchased through conventional commercial channels.

The toxic effect of cyanidin (Cyanidin Chloride) of experimental example 1 different concentrations to HASMC

Use Cell Counting Kit 8 (CCK-8; Dojindo Co, Kumamoto, Japan) to detect chlorinated cyanidins [molecular formula: C ₁₅ H ₁₁ ClO ₆ , purchased from Sigma-Aldrich], for HSAMC (purchased Cytotoxicity in ATCC Company) First, the 5th generation HSAMC was transferred to a 96-well plate (5×10 ³ cells per square centimeter), and cyanidin chloride was prepared in DMSO solution and added to a 10% fetal bovine Release different concentrations of cyanidin solutions (0, 12.5, 25, 50 and 100μM) in the high-glucose medium of serum, then add 100uL chlorinated cyanidin solution to each well, and treat the cells for 24 hours and 48 hours respectively . And set blank group (no cell without drug, only add 100uL serum medium) and control group (add equal amount of cells, no drug), when the cells grow to 80% of the 96-well plate, add phosphate buffered saline (PBS ), and then add 10 μL of CCK-8 and incubate at 37° C. for 4 h. Finally, the absorbance of the wells was measured at 450 nm using a microplate reader, all experiments were repeated three times, and the cell viability was calculated according to the absorbance:

Cell viability=(OD value of the drug treatment group-OD value of the blank group)/(OD value of the control group-OD value of the blank group)×100%, and Graphpad software was used for drawing and statistical results.

The results are shown in Figure 1, 50uM cyanidin chloride has the best activity on HSAMC at 24 hours, and has no toxic effect on HSAMC at 48 hours.

The mitigation effect of embodiment 2 cyanidin to HASMC calcification

HSAMC was seeded in a 12-well plate at passage 6, and calcification was induced when the cell growth reached 80% density. While adding high pi solution (2.8mM), 0, 12.5, 25, 50 and 100μM chlorine were added respectively. Calcium cyanidin was induced for a total of 7 days, and the medium was replaced every 2 days, and corresponding intervention treatments were added. After 7 days of calcification induction, alizarin red staining was performed, and the culture medium in the culture dish was sucked out, and PBS was gently Rinse three times, fix with 4% paraformaldehyde for 30min, then gently rinse with PBS three times, after absorbing the PBS, add 100uL of Alizarin Red staining solution to each well, stain for about 20 minutes, absorb the staining solution, rinse gently with distilled water for three times, and finally Microscopic observation and photographs were carried out, and the results are shown in Figure 2.

The results in Figure 2 prove that cyanidin chloride has a relieving effect on human vascular smooth muscle cells (human vascular smooth muscle cells, HASMC) that induces calcification, and the calcification staining is red, and among them, 12.5 μ M began to appear obvious relieving effect, 25 ,50μM, the relief effect is more significant.

Example 3 The preventive effect of cyanidin on high phosphorus (Pi) diet-induced calcification mice

(1) Establishment of CKD vascular calcification model in mice

Establishment of chronic kidney disease model in mice induced by high phosphorus and high adenine diet: 6-8 weeks old wild-type C57BL/6J male mice (purchased from Animal Experiment Center of Sun Yat-Sen University) were selected. The CKD vascular calcification model (belonging to endometrial calcification) was constructed by feeding with a special feed containing high phosphorus and high adenine containing 0.2% adenine and 1.2% phosphoric acid, and the total feeding time was 12 weeks.

(2) Experimental method

1) Effect of cyanidin on body weight of mice with calcification induced by high phosphorus diet

36 C57BL/6J male mice weighing 23-25g were randomly divided into 3 groups, 12 in each group, normal diet + sodium chloride (5mg/kg) tail vein injection group (ND), high phosphorus and high adenine + sodium chloride (5mg/kg) tail vein injection group (AP), and high phosphorus high adenine + chloride cyanidin (5mg/kg) injection group (cyanidin). Among them, the tail vein treatment of chlorinated cyanidin was started from the second week of the calcification induction model, and then the body weight changes of the mice were recorded every week, and the results were expressed in a broken line graph (see Figure 3)

2) Effect of cyanidin on the gross aorta of mice with calcification induced by high phosphorus diet

After 12 weeks of induction of calcification, the aorta of the mice was grossly dissected. After deep anesthesia, a median longitudinal incision was made on the abdominal wall to expose the abdominal cavity, so that the aorta was clearly exposed. The aorta was ligated at the aortic arch, and the disposable lancet was used to perfuse and gently flush with normal saline. After removing the remaining blood, the entire aorta was removed.

Mouse aortic tissues were fixed with 95% ethanol for 24 hours and stained with 0.003% Alizarin Red (dissolved in 1% sodium hydroxide) for 30 hours. Then the aorta was washed twice with 2% sodium hydroxide, and the staining results are shown in Figure 4.

(3) Experimental results

Figure 3 shows that after the tail vein treatment with cyanidin chloride from the 2nd week of the calcification-induced model, the body weight of the calcified mice began to show an obvious upward trend from the 9th week, indicating that cyanidin chloride can relieve the small Rats further developed vascular calcification.

Figure 4 shows that after the tail vein treatment with cyanidin chloride, the vascular calcification of the calcification-induced model mice was significantly improved, and the vascular calcification was effectively improved. It can be seen that cyanidin chloride has a preventive effect on mice with calcification induced by high phosphorus diet.

The comprehensive implementation of 1-3 shows that cyanidin (chlorinated cyanidin) can relieve vascular calcification, and is expected to be applied to the prevention and treatment of vascular calcification diseases.

The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions and modifications to these embodiments still fall within the protection scope of the present invention.

Claims (5)

1. The application of cyanidin in preparing medicine for preventing and treating vascular calcification is provided.

2. The use according to claim 1, wherein the vascular calcification is intimal calcification.

3. The use according to claim 1, wherein the cyanidin is chlorinated cyanidin.

4. The use of cyanidin in the preparation of a medicament for alleviating vascular cell calcification.

5. The use of claim 4, wherein the vascular cells comprise vascular smooth muscle cells.

Images